Ultra high performance liquid chromatography (UHPLC) has developed over the last decade in response to the need for faster chromatography techniques that can handle the vast number of samples generated during drug discovery and development. Compared to HPLC, which has been the predominant technology used in laboratories across a wide range of technical fields, UHPLC is carried out with columns comprising particles of a smaller size and increased mobile phase flow rates. Typically the particles present in UHPLC columns will be less than 2 μm in size, e.g. 1.7 to 1.8 μm, and typically a pressure of 50-100 MPa is used to achieve high throughput. The effect of the smaller particles is to ensure analytes are still separated in the column despite their shorter residence time therein.
UHPLC is used to monitor the progress of chemical reactions, as well as physical or other transformations, e.g. dissolution studies, in a wide range of different contexts. Reactions may, for instance, be monitored by analysing the depletion of a starting material or the generation of a product. UHPLC is, however, particularly useful during drug development wherein huge numbers of reactions take place. During drug development, UHPLC may be used to monitor the progress of reactions during chemical syntheses, to monitor the stability of compounds to different conditions and to monitor the pharmacokinetic properties of drugs and their formulations. During the formulation stage of drug development, for example, UHPLC may be used to assess the properties of different formulations, e.g. dissolution rates in a variety of different conditions. For instance, a range of pharmaceutical formulations may be prepared with varying amounts of a set of excipients and/or varying excipients and the dissolution rates of each of the formulations determined. This is carried out by placing each formulation in a dissolution media and then monitoring the media for the presence of the drug. The monitoring is carried out by removing a sample of the liquid at regular intervals and analysing it by UHPLC to determine the amount of drug present therein. The data gathered, usually in replicate, from the different time points can then be used to generate an average dissolution curve.
In a conventional UHPLC set up a rack for holding sample-containing vials is provided on a shuttle tray which is positioned in the UHPLC analyser wherein an aliquot of sample is taken from a first vial and analysed. The UHPLC analyser will then take an aliquot of sample from another vial in the array and analyse it. This process is repeated until a sample is taken from each vial and is analysed.
Typically, however, the manner of collecting and analysing samples from multiple vessels is very intensive, particularly if replicates of each sample are to be analysed. This burden is further exacerbated in the case of monitoring chemical reactions, physical or other transformations (hereinafter referred to collectively as “reaction monitoring” or “monitoring of reactions”), e.g. dissolution studies, wherein there is a need for the analysis to be repeated at a number of different points in time. Overall it means there is a need to collect a vast number of samples into vials and for these to be transported reliably and efficiently to the UHPLC analyser for analysis.
One way in which this problem has been overcome is to carry out the reaction, test or study, e.g. dissolution study, to be monitored in sample vials within a liquid chromatography device. This solution is, however, very limited. It is often not possible to set up reactions, tests or studies under the necessary conditions within the confines of a liquid chromatography device, e.g. it is not possible to provide any heating or cooling to the reaction, test or study within the device. It is also not possible to carry out reactions, tests or studies at anything other than a very small scale. Thus, for example, it is not possible to monitor the dissolution rates of many tablet formulations since the capacity of even the largest sample vials is too small to contain the required amount of dissolution media.
A need therefore still exists for improved equipment and methods for automated and continuous monitoring of reactions.